Affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

ABSTRACTOne of the major health risks to astronauts is radiation on long-duration space missions. Space radiation from sun and galactic cosmic rays consists primarily of 85% protons, 14% helium nuclei and 1% high-energy high-charge (HZE) particles, such as oxygen (16O), carbon, silicon, and iron ions. HZE particles exhibit dense linear tracks of ionization associated with clustered DNA damage and often high relative biological effectiveness (RBE). Therefore, new knowledge of risks from HZE particle exposures must be obtained. In the present study, we investigated the acute effects of low doses of 16O irradiation on the hematopoietic system. Specifically, we exposed C57BL/6J mice to 0.1, 0.25 and 1.0 Gy whole body 16O (600 MeV/n) irradiation and examined the effects on peripheral blood (PB) cells, and bone marrow (BM) hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) at two weeks after the exposure. The results showed that the numbers of white blood cells, lymphocytes, monocytes, neutrophils and platelets were significantly decreased in PB after exposure to 1.0 Gy, but not to 0.1 or 0.25 Gy. However, both the frequency and number of HPCs and HSCs were reduced in a radiation dose-dependent manner in comparison to un-irradiated controls. Furthermore, HPCs and HSCs from irradiated mice exhibited a significant reduction in clonogenic function determined by the colony-forming and cobblestone area-forming cell assays. These acute adverse effects of 16O irradiation on HSCs coincided with an increased production of reactive oxygen species (ROS), enhanced cell cycle entry of quiescent HSCs, and increased DNA damage. However, none of the 16O exposures induced apoptosis in HSCs. These data suggest that exposure to low doses of 16O irradiation induces acute BM injury in a dose-dependent manner primarily via increasing ROS production, cell cycling, and DNA damage in HSCs. This finding may aid in developing novel strategies in the protection of the hematopoietic system from space radiation.

pone.0158097.g005: 16O TBI drives HSCs from quiescence into the cell cycle.Lin−cells were isolated from control (CTL) and irradiated (TBI) mice 2 weeks after 0.1, 0.25 and 1.0 Gy TBI. Cell cycle was measured by flow cytometry using Ki-67 and 7-AAD double staining in BM HPCs and HSCs from control and irradiated mice. (A-C) The percentages of G0, G1 and G2SM phases in BM HPCs, LSK cells and HSCs after TBI are presented as mean ± SD (n = 5). The distribution of cell cycle phases in HPCs, LSK cells and HSCs was analyzed by Chi-Square test as indicated by X2 (HPC, X2 = 19.084, p<0.05; LSK cells, X2 = 6.486, p<0.05; HSCs, X2 = 33.853, p<0.05). The statistical significance for the difference in each cell cycle phase between the control groups and irradiated groups is indicated by asterisks. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA analysis.

Mentions:
Increased ROS production can stimulate stem cell cycling and cause oxidative DNA damage, resulting in stem cell exhaustion and induction of stem cell senescence. To investigate the cell cycle status of HPCs and HSCs, Ki-67 and 7-AAD double staining was utilized (Fig 5). Overall, the distribution of cell cycle phases in irradiated HPCs, LSK cells and HSCs was significantly different from that in non-irradiated counterpart populations (HPCs, X2 = 19.084, p<0.05; LSK cells, X2 = 6.486, p<0.05; HSCs, X2 = 33.853, p<0.05). Intriguingly, in irradiated mice, higher numbers of HPCs were in G0 and G2SM phases and lower numbers were in the G1 phase compared to the cell cycle distribution of HPCs in sham-irradiated mice (Fig 5A). As expected, the increased production of ROS in irradiated HSCs was associated with a significant reduction in LSK and HSC quiescence (fewer G0 phase cells p<0.05 and more G1 and G2SM phase cells, p<0.05-p<0.001) in a dose-dependent manner, indicating that 16O irradiation stimulated LSK and HSC cycling and proliferation (Fig 5B and 5C). Increased cycling of HSCs after 16O irradiation may compensate for the decrease of HSCs but at the expense of HSC self-renewal, which can result in the damage to the hematopoietic system.

pone.0158097.g005: 16O TBI drives HSCs from quiescence into the cell cycle.Lin−cells were isolated from control (CTL) and irradiated (TBI) mice 2 weeks after 0.1, 0.25 and 1.0 Gy TBI. Cell cycle was measured by flow cytometry using Ki-67 and 7-AAD double staining in BM HPCs and HSCs from control and irradiated mice. (A-C) The percentages of G0, G1 and G2SM phases in BM HPCs, LSK cells and HSCs after TBI are presented as mean ± SD (n = 5). The distribution of cell cycle phases in HPCs, LSK cells and HSCs was analyzed by Chi-Square test as indicated by X2 (HPC, X2 = 19.084, p<0.05; LSK cells, X2 = 6.486, p<0.05; HSCs, X2 = 33.853, p<0.05). The statistical significance for the difference in each cell cycle phase between the control groups and irradiated groups is indicated by asterisks. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA analysis.

Mentions:
Increased ROS production can stimulate stem cell cycling and cause oxidative DNA damage, resulting in stem cell exhaustion and induction of stem cell senescence. To investigate the cell cycle status of HPCs and HSCs, Ki-67 and 7-AAD double staining was utilized (Fig 5). Overall, the distribution of cell cycle phases in irradiated HPCs, LSK cells and HSCs was significantly different from that in non-irradiated counterpart populations (HPCs, X2 = 19.084, p<0.05; LSK cells, X2 = 6.486, p<0.05; HSCs, X2 = 33.853, p<0.05). Intriguingly, in irradiated mice, higher numbers of HPCs were in G0 and G2SM phases and lower numbers were in the G1 phase compared to the cell cycle distribution of HPCs in sham-irradiated mice (Fig 5A). As expected, the increased production of ROS in irradiated HSCs was associated with a significant reduction in LSK and HSC quiescence (fewer G0 phase cells p<0.05 and more G1 and G2SM phase cells, p<0.05-p<0.001) in a dose-dependent manner, indicating that 16O irradiation stimulated LSK and HSC cycling and proliferation (Fig 5B and 5C). Increased cycling of HSCs after 16O irradiation may compensate for the decrease of HSCs but at the expense of HSC self-renewal, which can result in the damage to the hematopoietic system.

Affiliation:
Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

ABSTRACTOne of the major health risks to astronauts is radiation on long-duration space missions. Space radiation from sun and galactic cosmic rays consists primarily of 85% protons, 14% helium nuclei and 1% high-energy high-charge (HZE) particles, such as oxygen (16O), carbon, silicon, and iron ions. HZE particles exhibit dense linear tracks of ionization associated with clustered DNA damage and often high relative biological effectiveness (RBE). Therefore, new knowledge of risks from HZE particle exposures must be obtained. In the present study, we investigated the acute effects of low doses of 16O irradiation on the hematopoietic system. Specifically, we exposed C57BL/6J mice to 0.1, 0.25 and 1.0 Gy whole body 16O (600 MeV/n) irradiation and examined the effects on peripheral blood (PB) cells, and bone marrow (BM) hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) at two weeks after the exposure. The results showed that the numbers of white blood cells, lymphocytes, monocytes, neutrophils and platelets were significantly decreased in PB after exposure to 1.0 Gy, but not to 0.1 or 0.25 Gy. However, both the frequency and number of HPCs and HSCs were reduced in a radiation dose-dependent manner in comparison to un-irradiated controls. Furthermore, HPCs and HSCs from irradiated mice exhibited a significant reduction in clonogenic function determined by the colony-forming and cobblestone area-forming cell assays. These acute adverse effects of 16O irradiation on HSCs coincided with an increased production of reactive oxygen species (ROS), enhanced cell cycle entry of quiescent HSCs, and increased DNA damage. However, none of the 16O exposures induced apoptosis in HSCs. These data suggest that exposure to low doses of 16O irradiation induces acute BM injury in a dose-dependent manner primarily via increasing ROS production, cell cycling, and DNA damage in HSCs. This finding may aid in developing novel strategies in the protection of the hematopoietic system from space radiation.